Project Summary Nitroxide spin labels are useful probes for investigation of biomacromolecules such as proteins DNA, and RNA. In a typical approach, doubly-labeled proteins are prepared by site-directed spin labeling (SDSL) and a set of distances between spin labels is measured using pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS). Double electron electron resonance (DEER) is the most widely used PDS method. The temperatures at which the DEER measurements currently can be made are limited by the dynamic averaging effects associated with methyl group rotation in the typical nitroxide spin label, and therefore have to be performed at about 50 ? 70 K, requiring use of liquid helium, or with lower sensitivity at 80 K with liquid nitrogen. In addition, application of the SDSL methodology to in vivo measurements is hampered by the short in vivo lifetime of the currently available nitroxide spin labels. The objective of the proposed work is to synthesize and evaluate new nitroxide spin labels for DEER distance measurements in proteins at physiological temperature and for in vivo DEER at cryogenic temperature. The proposed project has three specific aims. Aim 1: synthesize ultra-rigid, small-sized, and polar nitroxide spin labels for distance measurements at physiological temperatures (D-Labels). The target labels are novel nitroxides devoid of methyl groups with structure motifs that address the problem of conformational flexibility, molecular size and hydrophobicity. Aim 2: synthesize nitroxide spin labels for in vivo DEER distance measurements (C-Labels). The targets labels are biostable gem-diethyl and gem-dicarboxylate nitroxide spin labels. Aim 3: evaluate effectiveness of the synthesized spin labels for SDSL-DEER distance measurements at physiological temperatures and for in vivo DEER distance measurements. All labels synthesized in Aims 1 and 2 will be tested for their electron spin relaxation times (Tm and T1) as a function of temperature up to 310 K and for the rate of reduction in ascorbate/glutathione solutions. SDSL-DEER distance measurements at physiological temperature will be calibrated using T4 Lysozyme (T4L) that is doubly spin labelled with D-Labels and immobilized in carbohydrate matrix. C-Labels will be tested for in vivo DEER distance measurements at cryogenic temperature, via SDSL of selected proteins in live mitochondria and in E. coli. Collaborating laboratories will carry out these studies and set the stage for the general adoption of the proposed spin labels by highlighting their properties and defining the range of their applications. These studies will provide structural information at physiological temperature and/or in the native cellular environment, which is characterized by factors and conditions that may have a crucial role in determining the biologically relevant conformation of the proteins under investigation. The wealth of information enabled by the proposed labels will be close to ideal for understanding diseases and the approaches to developing cures for diseases.